The present invention relates generally to a computerized method and apparatus for analyzing sets of readings taken of respective samples in a biological or chemical assay, such as nucleic acid assay, to determine which samples possess a certain predetermined characteristic. More particularly, the present invention relates to a computerized method and apparatus which collects optical readings of a biological or chemical sample taken at different times during a reading period, corrects for abnormalities in the readings, and mathematically combines the values of certain of the readings to generate a sample indicator value which indicates a particular characteristic of the sample, such as the presence of a targeted pathogen in the sample.
In the clinical diagnosis of infectious disease, including sexually transmitted diseases such as Neisseria gonorrhea (GC) and chlamydia trachomatis (CT), a sample of body fluid obtained from the patient can be cultured to test for the presence of the particular organism of interest. Unfortunately, this is a relatively time-consuming process, generally requiring several days to produce a definitive result. During this time, a patient suspected of having such a disease may need to be isolated to prevent further spread of the disease.
The advent of DNA probes, which can identify specific organisms by testing for the presence of a unique DNA sequence in the sample obtained from the patient, has greatly increased the speed and reliability of clinical diagnostic testing. A test for the presence of CT or GC, for example, in a sample can be completed within several hours or less using DNA probe technology. This allows treatment to begin more quickly.
In the use of DNA probes for clinical diagnostic purposes, a nucleic acid amplification reaction is usually carried out in order to multiply the target nucleic acid into many copies or amplicons. Examples of nucleic acid amplification reactions include strand displacement amplification (SDA) and polymerase chain reaction (PCR). Detection of the nucleic acid amplicons can be carried out in several ways, most involving hybridization (binding) between the amplified target DNA and specific probes.
Many common DNA probe detection methods involve the use of fluorescein dyes. One known detection method is fluorescent energy transfer. In this method, a detector probe is labeled both with a fluorescein dye that emits light when excited by an outside source, and with a quencher which suppresses the emission of light from the flourescein dye in its native state. When DNA amplicons are present, the fluourescein-labeled probe binds to the amplicons, is extended, and allows fluorescent emission to occur. The increase of fluorescence is taken as an indication that the disease-causing organism is present in the patient sample.
Several types of optical readers or scanners exist which are capable of exciting fluid samples with light, and then detecting any light that is generated by the fluid samples in response to the excitation. For example, an X-Y plate scanning apparatus, such as the CytoFluor Series 4000 made by PerSeptive Biosystems, is capable of scanning a plurality of fluid samples stored in an array of microwells. The apparatus includes a scanning head for emitting light toward a particular sample, and for detecting light generated from the sample. During operation, the optical head is moved to a suitable position with respect to one of the sample wells. A light emitting device is activated to transmit light through the optical head toward the sample well. If the fluid sample in the well fluoresces in response to the emitted light, the fluorescent light is received by the scanning head and transmitted to an optical detector. The detected light is converted by the optical detector into an electrical signal, the magnitude of which is indicative of the intensity of the detected light. This electrical signal is processed by a computer to determine whether the target DNA is present or absent in the fluid sample based on the magnitude of the electrical signal. Each well in the microwell tray (e.g., 96 microwells total) can be read in this manner.
Another more efficient and versatile sample well reading apparatus is described in the above-referenced copending U.S. patent application, Ser. No. 08/929,895. In that system, a microwell array, such as the standard microwell array having 12 columns of eight microwells each (96 microwells total), is placed in a movable stage which is driven past a scanning bar. The scanning bar includes eight light emitting/detecting ports that are spaced from each other at a distance substantially corresponding to the distance at which the microwells in each column are spaced from each other. Hence, an entire column of sample microwells can be read with each movement of the stage.
As described in more detail below, the stage is moved back and forth over the light sensing bar, so that a plurality of readings of each sample microwell are taken at desired intervals. In one example, readings of each microwell are taken at one-minute intervals for a period of one hour. Accordingly, 60 readings of each microwell are taken during a well reading period. These readings are then used to determine which samples contain the particular targeted disease or diseases (e.g., CT and/or GC).
Several methods are known for analyzing the sample well reading data to determine whether a sample contained in the sample well includes the targeted disease or diseases. For instance, as discussed above, a nucleic acid amplification reaction will cause the target nucleic acid (e.g., CT or GC) to multiply into many amplicons. The fluorescein-labeled probe which binds to the amplicons will fluoresce when excited with light. As the number of amplicons increases over time while the nucleic acid amplification reaction progresses, the amount of fluorescence correspondingly increases. Accordingly, after a predetermined period of time has elapsed (e.g. 1 hour), the magnitude of fluorescence emission from a sample having the targeted disease (a positive sample) is much greater then the magnitude of fluorescence emission from a sample not having the targeted disease (a negative sample). In actuality, the magnitude of fluorescence of a negative sample essentially does not change throughout the duration of the test.
Therefore, the value of the last reading taken for each sample can be compared with a known threshold value, which has previously been determined. If the sample value is above the threshold value, the sample is identified as a positive sample in which the targeted disease is present. However, if the last value taken of the sample is below the threshold value, the sample is identified as a negative sample free from the disease.
Although this "endpoint detection" method can generally be effective in identifying positive and negative samples, it is not uncommon for this method to incorrectly identify a negative sample as being positive or vice-versa The accuracy of the value of any individual sample reading can be adversely effected by factors such as a bubble forming in the sample, obstruction of excitation light and/or fluorescence emission from the sample due to the presence of debris on the optical reader, and so on. Accordingly, if the final reading of a particular sample is erroneous and only that reading is analyzed, the likelihood of obtaining a false positive or false negative result is relatively high.
Furthermore, in some instances, the magnitude of fluorescent emission from the sample decreases with the passage of time due to, for example, quenching from the patient sample, side reactions from contaminants, or destruction of the fluorescein products due to unknown effects. Accordingly, the magnitude of the last reading taken of the sample can be less than the magnitude of a reading taken at the time when the signal from the sample is at its peak. In some instances, the signal can be lower then the predetermined threshold value, in which event the positive sample is falsely identified as a negative sample.
In order to avoid these drawbacks, other methods have been developed. In one method, the overall change in the magnitudes of sample readings is calculated and compared to a known value having a magnitude indicative of a positive result. Accordingly, if the magnitude of change is greater than the predetermined value, the sample is identified as a positive sample having the targeted disease. On the other hand, if the magnitude of change is less than the predetermined value, the sample is identified as a negative sample.
Although this method may be more effective than the endpoint detection method discussed above, certain flaws in this method also exist. For example, if a sample contains a particularly large amount of target DNA, the amount of amplicons generated due to the amplification process may reach a maximum at the time the initial reading is taken, and increase very little, if at all, or even decrease, throughout the duration of the reading period. In this event, the change which occurs between the initial readings and final readings is minimal even though the sample is positive. Hence, the sample may incorrectly be identified as a negative sample.
Accordingly, a continuing need exist for a method and apparatus for analyzing data representative of readings taken of sample wells to accurately identify the samples as being positive or negative for a particular disease.